Endogenous retroviral sequences are present as an integral part of eukaryotic genomes. Although the majority of these sequences are defective, a few can produce infectious virus, either spontaneously upon long-term culture or by treatment with various chemical or other agents. Early, extensive studies of retrovirus induction were done in mouse cells; however, similar studies have not been done using state-of-the-art virus detection assays and with cells of other mammalian species. To investigate induction and detection of occult retroviruses in cells of different species, especially primate cells that are used in production of biologics, we have initially determined the optimum conditions for retrovirus induction in chemically treated K-BALB mouse cells using highly sensitive product-enhanced reverse transcriptase (PERT) assays as well as transmission electron microscopy (TEM). Retrovirus induction was detected at day 1 post-drug treatment under all test conditions but was optimum using 30 microg ml(-1) of 5-iododeoxyuridine (IdU) for 24 h. Additionally, the combination of IdU and 5-azacytidine specifically enhanced activation of type C particles. RT activity was detected by PERT assays in one microliter equivalent of test sample and retroviral particle production was seen by TEM analysis. The induction of infectious murine leukemia retroviruses was confirmed by infectivity assays and correlated with PERT activity. These results indicate that strategies for detection of occult viral agents should include optimization of induction conditions using multiple viral detection assays to evaluate virus activation.